This application is a continuation-in-part of U.S. patent application Ser. No. 09/833,222 filed Apr. 11, 2001 and entitled “cDNA Encoding the Human Alpha2 Delta4 Calcium Channel Subunit”. The contents of which are hereby incorporated by reference in its entirety.
Voltage gated calcium channels (VGCC) mediate Ca2+ entry into cells in response to membrane depolarization (Catterall, W. A.(1988) Science 242:50-61 and Bean B P. (1989) Annu. Rev. Physiol. 51:367-368). Ca2+ entering the cell through voltage-gated Ca2+ channels serves as the second messenger of electrical signaling, initiating intracellular events such as contraction, secretion, synaptic transmission, and gene expression. Therefore, the VGCCs are involved in a variety of physiological processes in vertebrates, such as muscle contraction, insulin release from the pancreas, and neurotransmitter release in the nervous system (see Greenberg (1997), Annals of Neurology, 42:275-82; Rorsman et al. (1994), Diabete Metab. 20:138-145; and Catterall (1993), Trends in Neurosciences 16:500-506).
Electrophysiological studies reveal different types of calcium channels designated L-type (for Long Lasting), T-type (for Transient), N-type (for neither L nor T, or for “Neuronal”), P-type (for Purkinje cell), Q-type and R-type (for resistant) see Hess, (1990), Ann. NY Acad. Sci. 560:27-38; Bertolino and Llinás, (1992) Annu. Rev. Pharmacol. Toxicol. 32:399-421; and Randall and Tsien, (1995) J. Neurosci. 15:2995-3012). Except for the T type calcium channel, which is low voltage activated (LVA), the L-, N-, P-, Q- and R-types are all high voltage activated (HVA), i.e. their activation thresholds are normally above −40 mV.
The HVA Ca2+ channels that have been characterized biochemically are complexes of a pore-forming α1 subunit; a transmembrane, disulfide-linked complex of α2 and δ subunits; an intracellular β subunit; and in some cases a transmembrane γ subunit. To date, molecular cloning of calcium channels has revealed that there are ten α1 subunits, four α2δ complexes, four β subunits, and two γ subunits (Catterall, (2000), Annu. Rev. Cell Dev. Biol. 16: 521-555, and references therein). Analyses of these sequences indicate that the primary sequences of the calcium channel cDNAs have homologies ranging from between 40%-70%.
The primary structures of the five Ca2+ channel subunits were determined by combination of protein chemistry with cDNA cloning and sequencing. The α1 subunit is a protein of about 2000 amino acid residues with an amino acid sequence and predicted transmembrane structure like the previously characterized, pore-forming α subunit of the Na+ channels (Tanabe et al. (1987) Nature 328:313-18). The amino acid sequence is organized in four repeated domains (I to IV), each of which contains six transmembrane segments (S1 to S6) along with a membrane-associated loop between transmembrane segments S5 and S6. The intracellular β subunit has predicted α helices but no transmembrane segments (Ruth et al., (1989) Science 245:1115-18). The γ subunit is a glycoprotein with four transmembrane segments (Jay et al. (1990) Science 248:490-92). The cloned α2 subunit has many glycosylation sites and several hydrophobic sequences (Ellis et al. (1988) Science 241:1661-64), but biosynthesis studies indicate that it is an extracellular, extrinsic membrane protein attached to the membrane through disulfide linkage to the δ subunit (Gurnett et al (1996) Neuron 16:431-40). The δ subunit is encoded by the 3′ end of the coding sequence of the same gene as the α2 subunit. The mature forms of the α2 and δ subunits are produced by post-translational proteolytic processing and disulfide linkage (De Jongh et al (1990) J. Biol. Chem. 265:14738-41; and Jay et al (1991) J. Biol. Chem. 266:3287-93).
The α2δ subunit regulates most of the properties of the calcium channels, including voltage dependent kinetics and ligand binding (Qin et al, (1998) Am J. Physiol. (Cell Physiol.), 274: C1324-31). Altered α2δ expression is implicated in various disorders or diseases, such as epilepsy and other seizure-related syndromes, migraine, ataxia and other vestibular defects (for review see Terwindt et. al. (1998), Eur J Hum Genet 6(4):297-307), chronic pain (Backonja (1998), JAMA, 280(21):1831-6), mood, sleep interference (Rowbotham (1998), JAMA, 280(21):1837-42), anxiety (Singh et al. (1996), Psychopharmocology, 127(I): 1-9), ALS (Mazzini L et. al. (1998), J Neurol Sci 160 Suppl LS57-63), multiple sclerosis (Metz, (1998), Semin Neurol, 18(3):389-95), mania (Erfurth et al. (1998), J Psychiatr Res, 32(5):261-4), tremor (Evidente, et al. (1998), Mov Disord, 13(5):829-31), parkinsonism (Olson et al. (1997), Am J Med, 102(I):60-6), substance abuse/addiction syndromes (Watson et al. (1997), Neuropharmacology, 36(10):1369-75), depression, and cancer and at least one α2δ gene is located in a region of the genome which is thought to harbor an important tumor suppressor gene (Kok et al. (1997), Adv Cancer Res, 71:27-92). The defective α2δ gene has also been associated with proliferative diseases other than cancer, such as inflammation.
Characterizing the effects of the calcium channel subunit on ligand binding demonstrated that the α2δ subunit alters the binding of neurological and cardiovascular drugs to the ion channel pore-forming α1 subunit. Recently, gabapentin, a novel anticonvulsant drug, was shown to bind with high affinity directly to the calcium channel α2δ subunit (Gee, et al. (1996) J. Biol. Chem. 271:5768-76). Gabapentin may control neuronal excitability by modifying calcium channel activity or expression (Rock et al, (1993) Epilepsy Res. 16:89-98). More interestingly, antibodies directed against the α2δ subunit block secretion from PC12 cells, suggesting that the α2δ subunit may play a distinct role in neurotransmitter release (Gilad et al (1995) Neurosci. Lett. 193:157-60; Tokumaru, et al. (1995) J. Neurochem. 65:831-836 and Wiser, et al. (1996) FEBS Lett. 379:15-20). Further more, treatment with compounds that bind to α2δ leads to changes in the signal transduction mechanism of certain proteins including altered levels of MEK (MAP kinase kinase), an enzyme that activates the MAP kinase (mitogen-activated protein kinase). Activation of MAP kinase appears to be essential for cell proliferation and constitutive activation of this kinase is sufficient to induce cellular transformation.
An understanding of the pharmacology of compounds that interact with calcium channels and the design of such compounds is limited by an understanding of the genes that code for them. The identification of calcium channel subunits enables recombinant production of sufficient quantities of highly purified channel subunits which can be used in screening assays to identify or determine the effect of various compounds on channel function, thereby providing a basis for the design of therapeutic agents which affect the calcium channel. In particular, the identification of new α2δ subunits could present further possibilities for differential and specific regulation of calcium channels.